CFHR5 Antibody, FITC conjugated

What is CFHR5 and why is it an important research target?

CFHR5 (Complement Factor H-Related Protein 5) is a critical component of the complement regulation system. The protein is involved in complement regulation, with dimerized forms showing avidity for tissue-bound complement fragments. These forms efficiently compete with the physiological complement inhibitor CFH . CFHR5 co-localizes with C3, binds C3b in a dose-dependent manner, and is recruited to tissues damaged by C-reactive protein .

The protein contains nine short consensus repeats (SCRs), with the first two repeats having heparin binding properties, a region within repeats 5-7 having both heparin and C-reactive protein binding properties, and C-terminal repeats similar to a complement component 3b (C3b) binding domain . Allelic variations in CFHR5 have been associated with kidney diseases including membranoproliferative glomerulonephritis type II (MPGNII) and hemolytic uraemic syndrome (HUS) , making it an important target for research into complement-mediated renal pathologies.

What are the key specifications of commercially available CFHR5 Antibody, FITC conjugated?

Commercially available CFHR5 Antibody, FITC conjugated products typically have the following specifications:

These specifications are based on products described in multiple sources .

What are the recommended storage conditions for CFHR5 Antibody, FITC conjugated?

For optimal retention of activity, CFHR5 Antibody, FITC conjugated should be stored at -20°C or -80°C upon receipt . It is critically important to avoid repeated freeze-thaw cycles as these can degrade the antibody and diminish its fluorescence intensity. If aliquoting is necessary for long-term storage, divide the antibody into single-use volumes before freezing to minimize freeze-thaw events .

When working with the antibody, temporary storage at 2-8°C is acceptable for up to one month under sterile conditions after reconstitution . Always protect FITC-conjugated antibodies from light during storage and handling to prevent photobleaching, which can significantly reduce fluorescence signal intensity.

What applications is CFHR5 Antibody, FITC conjugated suitable for?

CFHR5 Antibody, FITC conjugated is suitable for several research applications that utilize fluorescent detection:

• Flow Cytometry (FACS): The FITC conjugation makes this antibody particularly suitable for flow cytometric analysis of cells expressing CFHR5 .

• Immunofluorescence Microscopy: Can be used for direct immunofluorescence visualization of CFHR5 in tissue sections or cellular preparations.

• Fluorescence-based Assays: May be used in various fluorescence-based detection methods.

How can I validate the specificity of CFHR5 Antibody, FITC conjugated in my experiments?

Validation of CFHR5 Antibody, FITC conjugated specificity is crucial for generating reliable research results. Based on experimental approaches used with similar antibodies, the following validation methods are recommended:

• Cross-reactivity Testing: Validate antibody specificity by testing against related proteins. For example, dot blot analysis with recombinant fragments of related proteins (CFHR1, CFHR2, CFHR3, CFHR4) demonstrated that certain CFHR5 antibodies do not cross-react with these related proteins .

• Positive and Negative Control Tissues/Cells: Use tissues known to express or not express CFHR5. Liver tissue is a positive control (as CFHR5 is synthesized in the liver), while heart tissue has been used as a negative control .

• Recombinant Protein Controls: Use recombinant CFHR5 protein fragments as positive controls .

• Knockout/Knockdown Validation: If available, cells or tissues with CFHR5 gene knockout or knockdown can provide definitive specificity validation.

• Pre-adsorption Tests: Pre-incubate the antibody with excess recombinant CFHR5 antigen before staining to demonstrate binding specificity.

• Western Blotting Verification: Even for FITC-conjugated antibodies intended for flow cytometry, verification of specificity by Western blot can be informative. CFHR5 typically appears as bands of approximately 64 kDa (de-glycosylated form) and 70-75 kDa (glycosylated form) .

How should I optimize the use of CFHR5 Antibody, FITC conjugated for flow cytometry?

Optimizing CFHR5 Antibody, FITC conjugated for flow cytometry requires attention to several methodological details:

• Titration Experiments: Determine the optimal antibody concentration by testing serial dilutions. Start with the manufacturer's recommended dilution and test 2-fold dilutions above and below this concentration .

• Buffer Optimization: Use appropriate buffers containing protein blockers (like 1-5% BSA or FBS) to reduce non-specific binding. The buffer composition can significantly impact staining quality.

• Controls: Include:

  • Unstained cells

  • Isotype control (FITC-conjugated rabbit IgG)

  • Positive control (cells/tissues known to express CFHR5)

  • Negative control (cells not expressing CFHR5)

  • Single-color controls if performing multicolor flow cytometry

• Fixation Considerations: If fixation is necessary, evaluate whether pre- or post-fixation staining yields better results. Note that some fixatives may affect the FITC fluorescence or epitope recognition.

• Compensation: When using multiple fluorophores, proper compensation is essential to account for spectral overlap. FITC has significant overlap with PE, so careful compensation is required when these fluorophores are used together.

• Signal Amplification: For low-abundance targets, consider signal amplification strategies like biotin-streptavidin systems if direct FITC detection proves insufficient.

• Instrument Settings: Optimize PMT voltages for the FITC channel (typically detected in FL1 on many cytometers) to ensure signal falls within the linear range of detection.

What are the advantages and limitations of using FITC-conjugated antibodies compared to other fluorescent conjugates for CFHR5 detection?

Advantages of FITC-conjugated CFHR5 antibodies:

• Well-established Fluorophore: FITC has been used extensively in immunofluorescence applications, with well-documented properties and detection parameters .

• Cost-effective: FITC is generally less expensive than newer generation fluorophores.

• Compatibility: FITC is compatible with most flow cytometers and fluorescence microscopes, utilizing standard 488 nm excitation laser lines .

• Direct Conjugation: Allows for direct one-step staining without the need for secondary antibodies.

Limitations of FITC-conjugated antibodies:

• Photobleaching: FITC is relatively prone to photobleaching compared to more photostable fluorophores like Alexa Fluor dyes.

• pH Sensitivity: FITC fluorescence is sensitive to pH changes, which may affect results in certain experimental conditions.

• Spectral Overlap: Significant overlap with other common fluorophores like PE can complicate multicolor experiments.

• Brightness: FITC is less bright than some newer fluorophores, which may impact detection of low-abundance targets.

• Autofluorescence Interference: FITC emission overlaps with cellular autofluorescence, potentially reducing signal-to-noise ratio, particularly in certain tissues like liver.

Alternative fluorophores to consider:

• Alexa Fluor 488 (similar spectral properties but greater photostability)

• PE (higher brightness for detecting low abundance antigens)

• APC (excitation/emission in the far-red range, reducing autofluorescence issues)

The choice between FITC and other fluorophores should be based on the specific research requirements, available instrumentation, and experimental design.

How does sample preparation affect the detection of CFHR5 using FITC-conjugated antibodies?

Sample preparation has significant impacts on the performance of CFHR5 Antibody, FITC conjugated across different experimental systems:

• Tissue Samples for Immunofluorescence:

  • Fixation method affects epitope accessibility. For CFHR5, paraformaldehyde fixation has been used successfully .

  • Antigen retrieval may be necessary - EDTA-based buffer at pH 8.0 has been effective for some CFHR5 antibodies .

  • Background autofluorescence can be problematic, especially in tissues like liver that naturally express CFHR5. Consider autofluorescence quenching methods.

• Cell Samples for Flow Cytometry:

  • Permeabilization is necessary for intracellular CFHR5 detection.

  • Cell viability can affect non-specific binding of antibodies. Using viability dyes to exclude dead cells is recommended.

  • Blocking of Fc receptors may be necessary to reduce non-specific binding.

• Protein Samples:

  • CFHR5 exists in both glycosylated (~70 kDa) and deglycosylated (~64 kDa) forms . Treatment with deglycosylation enzymes may affect epitope recognition.

  • Plasma samples require careful preparation due to the presence of numerous related complement factors.

• Buffer Considerations:

  • The presence of preservatives like Proclin 300 in the antibody solution must be considered when performing cellular assays .

  • The glycerol content (often 50%) in antibody storage solutions needs to be diluted sufficiently for cellular applications .

What methodological approaches can I use to study CFHR5 in complement-related diseases using FITC-conjugated antibodies?

When investigating CFHR5 in complement-related diseases using FITC-conjugated antibodies, consider these methodological approaches:

• Co-localization Studies:

  • Use multi-color immunofluorescence to co-localize CFHR5 (using FITC-conjugated antibodies) with other complement components like C3b or disease-specific markers.

  • This approach has revealed that CFHR5 co-localizes with C3 and is recruited to tissues damaged by C-reactive protein .

• Patient Sample Analysis:

  • Flow cytometric analysis of CFHR5 expression in blood cells from patients with complement-related disorders compared to healthy controls.

  • Analysis of CFHR5 binding to patient-derived samples can provide insights into disease mechanisms.

  • CFHR5 levels have been studied in patients with immune complex-mediated membranoproliferative glomerulonephritis and C3-glomerulopathy .

• Functional Competition Assays:

  • Develop assays to study how CFHR5 competes with factor H for binding to tissue-bound complement fragments .

  • Use FITC-labeled CFHR5 antibodies to track CFHR5 binding in these competitive scenarios.

• Tissue Deposition Patterns:

  • Map CFHR5 deposition patterns in diseased tissues (e.g., kidney biopsies from patients with C3 glomerulopathy).

  • Compare with patterns of other complement components to understand disease mechanisms.

• Mutant Protein Studies:

  • Investigate how disease-associated CFHR5 variants behave differently from wild-type protein using recombinant proteins.

  • FITC-conjugated antibodies can help track variant proteins in cellular systems.

• Therapeutic Intervention Models:

  • Use FITC-conjugated CFHR5 antibodies to monitor the effects of complement-targeting therapeutic interventions on CFHR5 levels or localization.

What are common issues encountered when using CFHR5 Antibody, FITC conjugated and how can I address them?

Researchers commonly encounter several issues when working with CFHR5 Antibody, FITC conjugated. Here are methodological solutions to these problems:

• Weak or No Signal:

  • Cause: Insufficient antibody concentration, epitope masking, or protein degradation

  • Solutions:

    • Titrate antibody to determine optimal concentration

    • Try alternative fixation methods that better preserve epitopes

    • Verify antibody activity using positive control samples

    • Check storage conditions - improper storage may have compromised fluorophore activity

• High Background:

  • Cause: Non-specific binding, autofluorescence, or insufficient blocking

  • Solutions:

    • Increase blocking time/concentration (5% normal serum from the same species as secondary antibody)

    • Include 0.1-0.3% Triton X-100 in blocking buffer to reduce non-specific interactions

    • Use tissue-specific autofluorescence quenchers

    • Ensure proper washing between steps with PBS containing 0.05-0.1% Tween-20

• Cross-Reactivity Issues:

  • Cause: Antibody binding to related complement factor H family proteins

  • Solutions:

    • Validate antibody specificity using dot blot analysis with recombinant CFHR proteins as demonstrated for some CFHR5 antibodies

    • Use competitive binding assays with unlabeled antibody or purified antigen

• Photobleaching:

  • Cause: FITC is susceptible to photobleaching during extended exposure

  • Solutions:

    • Minimize exposure to light during all preparation steps

    • Use anti-fade mounting media for microscopy

    • Capture images quickly or use lower excitation intensity

    • Consider alternative more photostable fluorophores for extended imaging

• Inconsistent Results Between Experiments:

  • Cause: Lot-to-lot variability, inconsistent sample preparation

  • Solutions:

    • Use the same antibody lot for related experiments when possible

    • Standardize all protocols including fixation, permeabilization, and staining times

    • Include standardized positive controls in each experiment

How can I optimize multiplex assays incorporating CFHR5 Antibody, FITC conjugated?

Optimizing multiplex assays with CFHR5 Antibody, FITC conjugated requires careful planning and execution:

• Fluorophore Selection and Combinations:

  • FITC has excitation/emission peaks at approximately 499/515 nm

  • Pair with fluorophores that have minimal spectral overlap, such as:

    • APC (excitation/emission: ~650/660 nm)

    • PE-Cy7 (excitation/emission: ~480-565/767 nm)

    • Pacific Blue (excitation/emission: ~401/452 nm)

• Panel Design Considerations:

  • Place FITC on abundant targets, as it's less bright than some alternatives

  • Account for FITC's significant spectral overlap with PE

  • Ensure all antibodies are validated individually before combining

  • Consider use of spectral viewers/panel design tools available from flow cytometer manufacturers

• Compensation Controls:

  • Prepare single-color controls for each fluorophore

  • Use compensation beads for consistent signal intensity

  • Adjust compensation matrix to eliminate spillover between channels

• Sample Preparation Optimization:

  • Test fixation and permeabilization protocols that work for all targets

  • Sequence antibody staining appropriately if different incubation conditions are required

  • Ensure blocking steps are sufficient to prevent non-specific binding

• Instrument Setup:

  • Calibrate the flow cytometer using standardized beads

  • Adjust PMT voltages for optimal resolution of all fluorophores

  • Use application-specific settings for consistent results between experiments

• Analysis Strategies:

  • Apply appropriate gating strategies

  • Use fluorescence minus one (FMO) controls to assist with gate placement

  • Consider dimensionality reduction techniques (tSNE, UMAP) for complex datasets

What methodological approaches can help distinguish between different forms of CFHR5 protein?

CFHR5 exists in multiple forms that can be distinguished using specific methodological approaches:

• Distinguishing Glycosylated vs. Deglycosylated Forms:

  • CFHR5 typically appears as bands of approximately 64 kDa (deglycosylated form) and 70-75 kDa (glycosylated form)

  • Methodology: Treat samples with protein deglycosylation enzymes (e.g., Protein Deglycosylation Mix II) and compare protein migration patterns by Western blot

  • FITC-conjugated antibodies can be used in microscopy to visualize the different forms if combined with appropriate sample treatment

• Detecting Dimerized CFHR5:

  • Dimerized forms of CFHR5 have increased avidity for tissue-bound complement fragments

  • Methodology: Use non-reducing vs. reducing conditions in SDS-PAGE followed by Western blotting

  • For fluorescence techniques, co-immunoprecipitation followed by fluorescence detection can identify dimeric complexes

• Identifying CFHR5 in Biological Fluids:

  • CFHR5 circulates either free or bound to HDL

  • Methodology: Density gradient ultracentrifugation to separate lipoprotein fractions, followed by immunodetection using FITC-conjugated antibodies

  • Alternatively, immunoprecipitation with anti-HDL antibodies followed by CFHR5 detection

• Mapping CFHR5 Domains:

  • CFHR5 contains nine SCRs with different functional properties

  • Methodology: Use antibodies targeting different epitopes (e.g., N-terminal vs. central vs. C-terminal regions)

  • Compare binding patterns of antibodies targeting amino acids 203-231 vs. 374-569 vs. other regions

• Detecting CFHR5 Genetic Variants:

  • Allelic variations in CFHR5 have been associated with kidney diseases

  • Methodology: Combine genetic testing with immunodetection to correlate genotype with protein expression/localization

  • Use FITC-conjugated antibodies to visualize localization patterns of variant proteins

How can CFHR5 Antibody, FITC conjugated be used to study complement-mediated pathologies?

CFHR5 Antibody, FITC conjugated offers several methodological approaches for investigating complement-mediated pathologies:

• Renal Disease Studies:

  • CFHR5 has been implicated in C3 glomerulopathy and membranoproliferative glomerulonephritis

  • Methodology: Use FITC-conjugated CFHR5 antibodies to map protein deposition patterns in renal biopsies

  • Co-stain with markers of complement activation (C3b, C5b-9) to understand the relationship between CFHR5 and complement dysregulation

  • Flow cytometric analysis of kidney-infiltrating cells using FITC-CFHR5 antibodies can reveal cellular sources or binding partners

• C-Reactive Protein (CRP)-Mediated Inflammation:

  • CFHR5 is recruited to tissues damaged by C-reactive protein

  • Methodology: Use in vitro models of CRP-mediated damage combined with FITC-CFHR5 antibody staining to visualize recruitment dynamics

  • Time-course experiments can reveal the temporal relationship between CRP deposition and CFHR5 recruitment

• Complement Regulation Mechanistic Studies:

  • CFHR5 competes with factor H for binding to tissue-bound complement fragments

  • Methodology: Develop competition assays using differentially labeled antibodies against CFHR5 and factor H

  • FITC-conjugated CFHR5 antibodies can help track the protein's localization during complement activation and regulation

• Biomarker Development:

  • Flow cytometric quantification of CFHR5 levels or binding patterns may serve as biomarkers for complement-mediated diseases

  • Methodology: Standardize flow cytometry protocols using FITC-conjugated CFHR5 antibodies for potential diagnostic applications

• Therapeutic Target Validation:

  • As understanding of CFHR5's role in disease grows, it may emerge as a therapeutic target

  • Methodology: Use FITC-conjugated antibodies to screen for compounds that modify CFHR5 binding or function

What emerging research areas might benefit from CFHR5 Antibody, FITC conjugated studies?

Several emerging research areas stand to benefit from studies utilizing CFHR5 Antibody, FITC conjugated:

• Complement's Role in Neurodegenerative Diseases:

  • Methodology: Apply FITC-conjugated CFHR5 antibodies to study whether CFHR5 is involved in complement dysregulation in neurodegenerative conditions

  • Flow cytometric analysis of microglia and other CNS cells could reveal CFHR5 interactions in neuroinflammation

• COVID-19 and Complement Activation:

  • Recent research has highlighted complement's role in COVID-19 pathology

  • Methodology: Use FITC-CFHR5 antibodies to investigate whether CFHR5 is involved in complement activation in COVID-19 infected tissues

• Cancer Microenvironment Studies:

  • Complement activation in the tumor microenvironment affects tumor progression

  • Methodology: Apply FITC-CFHR5 antibodies in flow cytometry and imaging to explore CFHR5's role in tumor-related complement activation

• Extracellular Vesicle (EV) Research:

  • Complement proteins may associate with EVs in various disease states

  • Methodology: Use FITC-conjugated CFHR5 antibodies to detect CFHR5 on isolated EVs by flow cytometry or fluorescence correlation spectroscopy

• Single-Cell Analysis of Complement Dynamics:

  • Methodology: Incorporate FITC-CFHR5 antibodies in single-cell proteomics approaches

  • Combine with RNA sequencing in techniques like CITE-seq to correlate CFHR5 protein levels with transcriptional profiles

• Organ-on-a-Chip Models:

  • Microfluidic organ models offer controlled environments to study disease mechanisms

  • Methodology: Use FITC-CFHR5 antibodies for real-time imaging of complement dynamics in kidney-on-a-chip or other relevant models

How can I design experiments to elucidate the functional relationship between CFHR5 and other complement components?

To investigate functional relationships between CFHR5 and other complement components, consider these methodological approaches:

• Co-localization Studies:

  • Design: Multi-color immunofluorescence using FITC-conjugated CFHR5 antibodies and antibodies against other complement components (C3b, Factor H, C5b-9)

  • Analysis: Quantify co-localization coefficients (Pearson's, Manders') to determine the degree of spatial association

  • Applications: Tissue sections from disease models or cell culture systems exposed to complement activators

• Competitive Binding Assays:

  • Design: Develop solid-phase binding assays where purified CFHR5 competes with other complement regulators (e.g., Factor H) for binding to immobilized C3b

  • Detection: Use FITC-conjugated CFHR5 antibodies to quantify bound CFHR5 under various competitive conditions

  • Controls: Include dose-response curves and specificity controls

• Functional Complement Assays:

  • Design: Hemolysis assays or complement activation ELISAs in the presence of purified CFHR5, with or without other complement regulators

  • Analysis: Use FITC-conjugated CFHR5 antibodies to track CFHR5 during the assay by sampling at different time points

  • Variables: Test different CFHR5 concentrations, disease-associated CFHR5 variants, or modified proteins

• Protein-Protein Interaction Studies:

  • Design: Pull-down assays or co-immunoprecipitation experiments to identify CFHR5 binding partners

  • Detection: Use FITC-conjugated CFHR5 antibodies for direct visualization of interactions

  • Validation: Confirm interactions using complementary techniques like surface plasmon resonance

• Cellular Binding and Internalization:

  • Design: Expose cells (e.g., renal tubular cells, endothelial cells) to purified CFHR5 and track binding/internalization

  • Detection: Use FITC-conjugated CFHR5 antibodies for flow cytometry or live cell imaging

  • Competition: Add other complement components to determine how they affect CFHR5-cell interactions

• Genetic Manipulation Approaches:

  • Design: Use CRISPR/Cas9 to create CFHR5 knockout or knock-in cell lines

  • Analysis: Compare complement activation and regulation in modified vs. control cells using flow cytometry with FITC-conjugated antibodies

  • Rescue Experiments: Reintroduce wild-type or mutant CFHR5 to knockout cells to assess functional recovery

These experimental approaches provide a comprehensive framework for unraveling the complex functional relationships between CFHR5 and other components of the complement system in both normal physiology and disease states.

What key publications should I review before designing experiments with CFHR5 Antibody, FITC conjugated?

Before designing experiments with CFHR5 Antibody, FITC conjugated, researchers should review these seminal publications that provide critical background on CFHR5 biology and methodology:

• McRae JL, et al. (2001) "Identification of a Novel Complement Factor H-Related Protein in Human Plasma" - First description of CFHR5 protein and its characteristics.

• McRae JL, et al. (2005) "Human Factor H-Related Protein 5 Has Cofactor Activity, Inhibits C3 Convertase Activity, Binds Heparin and C-Reactive Protein, and Associates with Lipoprotein" - Landmark paper detailing CFHR5 functions and interactions .

• Garam N, et al. "FHR-5 Serum Levels and CFHR5 Genetic Variations in Patients With Immune Complex-Mediated Membranoproliferative Glomerulonephritis and C3-Glomerulopathy" - Important for understanding CFHR5's role in kidney diseases .

• Chen Q, et al. (2016) "Complement Factor H-Related 5-Hybrid Proteins Anchor Properdin and Activate Complement at Self-Surfaces" - Reveals novel mechanisms of CFHR5 in complement dysregulation .

• Trégouët DA, et al. publication in Nature Communications - Contains key information on CFHR5 analysis in human samples .

• Studies using western blot analysis showing CFHR5 molecular weight patterns and validation approaches for antibody specificity .

What methodological resources are available for optimizing CFHR5 detection protocols?

Several methodological resources can help researchers optimize CFHR5 detection protocols:

• Product-Specific Technical Resources:

  • Detailed protocols from antibody manufacturers, including recommended dilutions, buffer compositions, and application-specific guidelines

  • Technical support services offered by antibody suppliers for troubleshooting

• General Method Resources:

  • Flow cytometry optimization guides from core facilities and commercial providers

  • Immunofluorescence staining optimization handbooks

  • Online method repositories like Bio-protocol or JoVE for visualized protocols

• Validation Resources:

  • The International Working Group for Antibody Validation (IWGAV) guidelines

  • Antibody Validation Database resources for checking validation status

• Fluorophore Resources:

  • Spectral viewers to plan multicolor experiments with FITC-conjugated antibodies

  • Fluorophore handbook resources detailing FITC properties, advantages, and limitations

• Application-Specific Publications:

  • Publications demonstrating dot blot analysis for specificity testing

  • Western blot protocols showing detection of both glycosylated (~70 kDa) and deglycosylated (~64 kDa) forms of CFHR5

  • Immunohistochemistry protocols using EDTA-based, pH 8.0 buffer for antigen retrieval

What quality control data should I expect when purchasing CFHR5 Antibody, FITC conjugated?

When purchasing CFHR5 Antibody, FITC conjugated, researchers should expect the following quality control data to be provided:

• Specificity Testing:

  • Evidence of specific binding to CFHR5 protein

  • Cross-reactivity testing against related proteins (e.g., CFHR1-4)

  • Lot-specific validation data

• Performance Testing:

  • Validation data in at least one application (e.g., flow cytometry, immunofluorescence)

  • Recommended dilution ranges based on testing

  • Example images or data showing expected results

• Physical Characterization:

  • Protein concentration measurement

  • Buffer composition details

  • Conjugation efficiency (FITC:protein ratio)

  • Appearance and physical state inspection

• Stability and Storage Information:

  • Expiration date

  • Storage requirements

  • Freeze-thaw stability data

• Additional Information:

  • Immunogen details (e.g., the specific sequence used to generate the antibody)

  • Host species and clonality confirmation

  • Isotype information

  • Purification method used